We had previously shown that the first nonstructural protein in the DEN2 genome, NS1, can trans complement lethal mutations of this protein in the full-length "infectious" cDNA clone. Defective virus produced in complementing, NS1-expressing cells can be passaged in NS1-expressing cells, but not in wt cells. We have also introduced a series of mutations in the carboxy-terminus of the NS1 gene sequence in the context of the DEN2 infectious cDNA to elucidate further NS1 function in virus replication. Cells expressing NS1 were also shown to enhance the replication of wt DEN virus by approximately 100-fold. We have single-cell cloned our NS1 expressing cell line and are selecting stable populations which express a high level of NS1 to optimize the enhancement of virus replication. This cell line will be used to study how NS1 functions in the virus life cycle and how NS1 working in trans contributes to the enhancement of virus replication. During flavivirus infections, NS1 is expressed at the surface of infected cells and secreted into the medium in vitro. It was originally identified as a "soluble complement-fixing antibody" in the sera of patients infected with dengue viruses. The immune response to NS1 can therefore be protective, by limiting cell-to-cell spread of virus, rather than by preventing infection. The immune response to NS1 is reactive across flavivirus species, unlike the immune response to the major viral structural antigen, E (envelope). Therefore, others have proposed that a single dengue virus vaccine containing NS1 could protect against all 4 serotypes. In contrast to the in-depth information available regarding the immune response to NS1 and its metabolism, nothing is known about NS1 function during virus replication. Results of this project to date suggest that experimental approaches to gaining such information for the first time are available. The finding that wt virus replication is enhanced in NS1-expressing cells may prove significant for the manufacture of a live virus vaccine. An invention report is being filed.